iPSCs were maintained in serum- and feeder-free conditions in Essential 8 Medium (E8, #A15169-01, Thermo Fisher Scientific) supplemented with 0.5% penicillin-streptomycin (P/S, #15140122 Thermo Fisher Scientific) on growth factor reduced Matrigel (Corning) at 5% CO2 and 37 °C. Medium was completely changed every 24 h. Twice a week, when cultures reached 80% confluency, the cells were passaged as small colonies with 0.5 mM EDTA (Thermo Fisher Scientific) in Ca2+/Mg2+-free DPBS (Thermo Fisher Scientific). The medium was supplemented with 5 µM of Y-27632 (#S1049, Selleckchem, Houston, TX, USA), a Rho-Associated Coil Kinase (ROCK) inhibitor (#S1049, Selleckchem,), for the first 24 h to prevent apoptosis after passaging. Cultures were tested for mycoplasma using a MycoAlert Kit (Lonza, Basel, Switzerland)), and for bacteria, mould, fungi and yeast using solid and liquid lysogeny broth (LB, Merck, KGaA, Darmstadt, Germany) and sabouraud dextrose agar (SDA, VWR, Radnor, PA, USA) at 30 °C and 37 °C.
Sabouraud dextrose agar
Manufactured by Avantor
Sourced in Germany, United States, Spain, United Kingdom, France
Sabouraud dextrose agar is a microbiological culture medium used for the isolation and cultivation of fungi and yeast. It is formulated with dextrose as a carbon source and provides a suitable environment for the growth of these microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Essential 8 medium e8, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Selleck Chemicals (1 mentions)
Mycoalert kit, by Lonza (1 mentions)
Growth factor reduced matrigel, by Corning (1 mentions)
Glycerol, by Avantor (1 mentions)
Potato dextrose agar (pda), by Biolife (1 mentions)
Reference antibacterial and antifungal compounds, by Merck Group (1 mentions)
Rpmi 1640, by Avantor (1 mentions)
Mueller hinton agar (mha), by Avantor (1 mentions)
Milli q reference, by Merck Group (1 mentions)
Mueller hinton broth (mhb), by Avantor (1 mentions)
Tryptic soy agar, by Avantor (1 mentions)
A castellanii, by American Type Culture Collection (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
6 protocols using sabouraud dextrose agar
1
Feeder-free iPSC Maintenance Protocol
2
Cultivation of Diverse Yeast Species
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Collection strains from the American Type Culture Collection (ATCC) and the German Collection of Microorganisms and Cell Cultures (DSMZ) were used, namely Candida albicans ATCC 10231, Rhodotorula mucilaginosa DSM 13621, Malassezia furfur DSM 6170, and Naganishia albida DSM 70215. The cultures were kept at −80 °C in Brain Heart Infusion broth (BHI, VWR, Avantor, Radnor, PA, USA) supplemented with 20% glycerol (VWR, Avantor, Radnor, PA, USA). Yeast cultures were inoculated on Sabouraud Dextrose Agar (SDA, VWR, Avantor, Radnor, PA, USA) plates (for C. albicans and R. mucilaginosa and incubated at 37 °C and 25 °C, respectively), on Potato Dextrose Agar (PDA, Biolife, Monza, Italy) plates (for N. albida and incubated at 25 °C), and on SDA with 0.5% olive oil and 0.5% (v/v) Tween-80 (SDA supplemented) for M. furfur at 37 °C.
3
Antioxidant, Enzyme Inhibition, and Antimicrobial Assays
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The chemical compounds used for the antioxidant methods, the reagents for LOX and AChE inhibition assays and reference antibacterial and antifungal compounds were purchased from Sigma-Aldrich (Madrid, Spain). All compounds were of analytical grade (purity higher than 95%). All culture media were acquired from VWR Chemicals (Barcelona, Spain): Mueller-Hinton agar (MHA), Mueller-Hinton broth (MHB), Roswell Park Memorial Institute medium (RPMI-1640), Sabouraud dextrose agar (SDA), tryptic soy broth (TSB) and yeast peptone dextrose (YPD). Solvents of analytic grade and buffers were purchased from Merck (Madrid, Spain). Type I (18 MΩ·cm) deionized water (MilliQ-Reference, Millipore, Madrid, Spain) was used in this work.
4
Culturing Pathogenic Microbes and Protists
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The S. aureus (NCTC 10788) and P. aeruginosa (NCTC 12924) strains used in this study were obtained from the National Collection of Type Cultures, Porton Down, UK. Both were cultured using tryptic soy agar (VWR Ltd., Leicestershire, UK) and incubated at 32 °C. The fungal pathogen C. albicans (NCPF 3179) was obtained from the National Collection of Pathogen Fungi, Porton Down, UK. This was cultured using sabouraud dextrose agar (VWR Ltd., Leicestershire, UK) and incubated at 32 °C. The Acanthamoeba strains, A. polyphaga (ATCC 30461) and A. castellanii (ATCC 50370), were obtained from the American Type Culture Collection (LGC Standards, Teddington, UK). Trophozoites were maintained in tissue culture flasks in Ac#6 medium at 30 °C in a static incubator, and cysts were produced using Neff’s encystment medium (NEM) in tissue culture flasks at 30 °C in a shaking incubator as previously described [7 (link)].
5
Identification of Azole-resistant Aspergillus
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Three clinical strains of Af, isolated from respiratory samples, were used in the present study. Identification was confirmed by sequencing part of the gene encoding beta-tubulin. The CYP51A gene and its promoter had been previously sequenced to determine the mutations involved in azole-resistance (Jemel et al., 2021 (link)). We included one azole-susceptible strain (AfS) with a wild type CYP51A sequence, one strain (AfR1) with a G54W mutation and one strain (AfR2) with a L98H point mutation in CYP51A in combination with a 34-bp tandem repeat in the promoter (TR34/L98H).
Subcultures were performed on Sabouraud dextrose agar (VWR, Fontenay-sous-bois, France) with chloramphenicol (Sigma-Aldrich, Saint Quentin-Fallavier, France). They were incubated for 7 days at 37°C to obtain sufficient sporulation.
Subcultures were performed on Sabouraud dextrose agar (VWR, Fontenay-sous-bois, France) with chloramphenicol (Sigma-Aldrich, Saint Quentin-Fallavier, France). They were incubated for 7 days at 37°C to obtain sufficient sporulation.
6
Fungal Pathogen Identification and Quantification
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The pathogen structures are located on almost one half of the leaf area isolated and cultured on medium -PDA or SA (Dehydrated culture media, potato dextrose agar by MP Biomedicals, Sabouraud dextrose agar by VWR, part of Avantor) to identify a particular species of pathogen. Cultivation on medium was performed under normal laboratory conditions for 7-10 days at laboratory temperature of 20/23 °C and light regime of 14/10 hours. Statistical data processing (monitoring of weeds and pathogens) was performed using multidimensional analysis of ecological data. Data was processed by DCA (Detrended Correspondence Analysis) to calculate the length of the gradient (Length of Gradient). Subsequently Canonical Correspondence Analysis (CCA) was used. This way the cover of the found weed species was evaluated, and the cover of annual ryegrass was used as a factor of environment. The intensity of infection of fungal pathogens was processed by multivariate analysis of ecological data. The day of evaluation was used as an intermediate factor. Subsequently, redundancy analysis (RDA) was used.
Statistical significance testing was performed using the Monte-Carlo test and 999 permutations were calculated. The calculations were performed using the computer program Canoco 5.0. (Ter Braak and Šmilauer, 2012) .
Statistical significance testing was performed using the Monte-Carlo test and 999 permutations were calculated. The calculations were performed using the computer program Canoco 5.0. (Ter Braak and Šmilauer, 2012) .
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